Oral dual-therapy for prevention of obesity

Obesity

is a highly prevalent metabolic disorder which is scientifically defined as an excessive, abnormal accumulation of adipose tissue. A total of 988 million people has been categorised as obese in 2020, which accounts for approximately 14% of the global population, depicting its pandemic proportion. It is also one of the most profound risk factors that contributes to increased risk associated with numerous fatal non-communicable diseases, including but not limited to diabetes mellitus type II, cardiovascular diseases, and cancer. Among the few existing pharmacological treatment options, many of them would cause serious adverse ramifications on users’ health.

Part 1 (Probiotics Team)

Farnesoid X receptor (FXR)

is a nuclear receptor that plays a crucial role in the regulation of bile acid, lipid, and glucose metabolism.

Firstly, FXR target certain growth factors. Activation of FXR upregulates FGF19, which is a growth factor proved to act on the liver to inhibit bile acid synthesis, promote glycogen synthesis, increase insulin sensitivity in peripheral tissues (Talukdar & Kharitonenkov, 2021), and also has effect on the brain to modulate appetite and energy expenditure so as to reduce food intake and increase energy expenditure (Stanley & Buettner, 2014). Apart from FGF19, FXR also targets FGF21 to induce weight loss by regulating fatty acid oxidation and improve glycemia (Fisher & Flier, 2016). Besides, it is proved to cause browning and proceed anti-inflammatory effect (Geng et al., 2020). On top of this, FGF21 is shown to associated with skeletal muscle and bone lost during aging, causing sarcopenia (Oost et al., 2019). In addition, it is also highly associated with obesity in elderly. It is suggested that muscle and bone mass decrease with aging, while fat mass increases (Colleluori & Villareal, 2021).

Lactobacillus casei

is chosen to be the vector, with target genes FGF15, FGF19, and FGF21 inserted. Not only is Lactobacillus casei the major bacterial community in small intestine, it is also the strain of Lactobacillus found in many dairy drinks such as Yakult (益力多), making it a perfect candidate for “oral drug”. Moreover, L. casei has a relatively versatile metabolism and can utilize a wide range of primary bile acid to produce secondary bile acids, the primary substrate to the FXR receptor. Therefore, we proposed, by cloning corresponding genes (separately) into Lactobacillus casei, it could acquire anti-obesity properties.

However, there are certain limitations shown in the natural occurring FGF proteins, such as poor pharmacokinetic and biophysical properties. Individuals with obesity might even develop systemic and/or adipose depot-selective FGF21 resistance and underpin insulin resistance (Geng et al., 2020). Modifications were therefore performed to improve their stability, half-life, membrane solubility or resistance against degradation. (Details are stated in Engineering)

Part 2 (miRNA Team)

A eukaryotic cell line that stably produces adipose-targeted exosome with anti-inflammation, pro-beiging, and anti-senescence effects for obesity prevention will be generated.

The genetic circuit

was created by inserting 3 siRNA sequences into respective 166-bp pre-miR-155 backbone, consisting of a CMV promoter and pre-miRNA parts. The CMV promoter was used to express a miRNA precursor, and the miRNA sequence was substituted with siRNA. Exosomes were chosen due to their relative lack of side effects compared to naked miRNAs/siRNA or other delivery vehicles. For the selection of siRNAs, we identified certain genes and knockdown targets. To start off, we will target the MKK6 gene which is elevated in the white adipose tissue of obese individuals. Literature has shown that the deletion of MKK6 can increase energy usage and thermogenic capacity of one’s white adipose tissue, helping to protect against further development of obesity. In addition, MKK6 deletion also stimulates T3-stimulated UCP1 expression in adipocytes. This also increases thermogenic capacity. Hence, the MKK6 gene holds potential to alleviate obesity. We will also target the p53 gene. The p53 gene can modulate lipid metabolism and studies have found that p53 activation in obese mice and humans promotes the senescence of adipocytes and the recruitment of macrophages. Finally, the p65 gene was chosen as due to its role in the NF-kappaB pathway, which could possibly induce inflammation significantly.

The HEK293 cells

are immortalized human embryonic kidney cells, commonly used in biomedical research and were chosen due to their high efficiency and stability for transfection. This plasmid will be co- transfected to the HEK293 cells. This process of transfection will go on for 48 hours. After this, the exosome will be produced and secreted into the cell supernatant. Once again, we will wait for 48-72 hours, after which, puromycin will be added. Puromycin is a naturally derived antibiotic that hinders the production of proteins by inserting itself into the end of growing protein chains, preventing their further elongation and causing the translation process to end prematurely. Puromycin is added to our cells mainly to screen for stable infections as puromycin resistant HEK293 cells contain the DNA construct permanently incorporated into the cell genome. Following this, the cells can now stably produce the adipose-targeted exosome with miRNAs of interest.

Finally, we will combine these projects to synthesize a weight loss yogurt – a dual-therapy for prevention of obesity.

References

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Colleluori, G., & Villareal, D. (2021). Aging, obesity, sarcopenia and the effect of diet and exercise intervention. Experimental Gerontology, 155. https://doi.org/10.1016/j.exger.2021.111561

Geng, L., Lam, K., & Xu, A. (2020). The therapeutic potential of FGF21 in metabolic diseases: from bench to clinic. Nature Reviews Endocrinology, 16, pages654-667. https://doi.org/10.1038/s41574-020-0386-0

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